(1) Field of the Invention
This invention concerns 2,6,9-trisubstituted purines that have been discovered to be selective inhibitors of cell cycle kinases and, as such, the compounds are inhibitors of cell proliferation. The 2,6,9-trisubstituted purines are useful in for example inxe2x80x94treating autoimmune diseases, e.g. rheumatoid arthritis, lupus, type I diabetes, multiple sclerosis, etc., in treating cancer, cardiovascular disease, such as restenosis, host vs graft disease, gout, polycystic kidney disease and other proliferative diseases whose pathogenesis involves abnormal cell proliferation.
This invention also concerns 2,6,9-trisubstituted purines that have been discovered to be potent and specific inhibitors of IxcexaB-xcex1 kinase which prevents signal induced NF-xcexaB activation and cytokine synthesis in vitro and in vivo. Such inhibitors are expected to inhibit the synthesis of cytokines and adhesion proteins whose synthesis is transcriptionally regulated by NF-xcexaB. Proinflammatory cytokines such as IL-1, IL6, TNF and adhesion proteins (e.g. ICAM, VCAM and selections) belong to this class of molecules and have been implicated in the pathogenesis of inflammatory diseases. Thus a potent inhibitor of IxcexaB-xcex1 kinase is useful in the clinical management of diseases where NF-xcexaB activation is required for disease induction.
(2) Description of the Art
In the past few years, advances in molecular and cellular biology have contributed to our understanding of the mechanisms of cell proliferation and of specific events that occur during progression of cells through mitosis. E.g., xe2x80x9cProgress in Cell Cycle Researchxe2x80x9d Vol 1, Eds. L. Meijer, S. Guidet and H. Y. L. Tung; Plenum Press, New York, 1995. These studies have shown that progression through the cell cycle is controlled by a family of serine/threonine kinases called cyclin dependent kinases. These enzymes contain (a) a catalytic protein called cyclin dependent kinase (CDK) that uses ATP as a substrate and (b) a regulatory protein called cyclin. Different cyclin-CDK combinations control events such as growth, DNA replication and cell division. One key member of the CDK family of enzymes is CDK2. CDK2 activity has been shown to be essential for mammalian cell cycle progression at the G1/S boundary. Microinjection of antibodies directed against CDK2 blocks the progression of human diploid fibroblasts into the S phase of the cell cycle. Expression of a CDK2dominant negative mutant in human osteosarcoma cells has a similar effect. Together, these studies indicate that inhibition of cellular CDK2 activity will prevent progression of cells through the mitotic cycle and induce growth arrest prior to the S phase. Consistent with this view, in vitro studies with olomoucine (2-(hydroxyethylamino)-6-benzylamino-9-methylpurine), have shown that it is a specific inhibitor of CDK2 with an IC50 of approximately 2.1 xcexcg/ml J. Vesely, et al.; Eur. J. Biochem 224, 771-786 (1994), L. Meijer xe2x80x9cChemical Inhibitors of Cyclin-Dependent Kinasesxe2x80x9d pp 351-356 in xe2x80x9cProgress in Cell Cycle Research Vol 1, Eds. L. Meijer, S. Guidet and H. Y. L. Tung; Plenum Press, New York, 1995. In vivo studies using mammalian cells in culture have shown that olomoucine inhibits cell proliferation at an approximate concentration of 50 xcexcg/ml.
In this invention, we have developed several compounds whose biological activity is considerably more potent than olomoucine. In vivo studies using mammalian cells indicate that some of the disclosed compounds inhibit cell proliferation at concentrations that are significantly lower than olomoucine.
Recently an IxcexaB-xcex1 kinase activity has been described in the cytoplasm of stimulated human umbilical vein endothelial cells (Bennett et al (1996) J. Biol. Chem 271, 19680-19688). Some of the compounds of this invention have been identified as potent and specific inhibitors of IxcexaB-xcex1 kinase which prevents signal induced NF-xcexaB activation and cytokine synthesis in vitro and in vivo. The activation of the heterodimeric transcription factor NF-xcexaB is a complex process. In unstimulated cells, the NF-xcexaB (p50/p65) heterodimer is located in the cytosol where it is complexed with an inhibitory subunit IxcexaB-xcex1, IxcexaB-xcex1, binds to NF-xcexaB thus masking its nuclear localization signal and preventing translocation to the nucleus. Upon stimulation of cells with a variety of signals (e.g. lipopolysaccharide) IxcexaB-xcex1 is rapidly phosphorylated, uniquitinated and degraded by the proteasome. Degradation of IxcexaB-xcex1, allows the translocation of NF-xcexaB to the nucleus where it activates transcription of a number of inflammatory response genes.
These observations suggest that IxcexaB-xcex1 kinase is an attractive target for the identification of inhibitors that may be useful in the treatment of inflammatory diseases where NF-xcexaB activation is required for disease induction.
It is an object of this invention to provide 2,6,9-trisubstituted purine compounds, which inhibit the cyclin dependent kinase 2.
It is another object of this invention to provide 2,6,9-trisubstituted purine compounds which are useful for inhibiting cell proliferation.
This invention also constitutes a pharmaceutical composition, which comprises a 2,6,9-trisubstituted purine compound and a pharmaceutically acceptable carrier.
This invention further constitutes a method for inhibiting cell proliferation, which comprises administering to a mammal in need thereof an effective amount of a 2,6,9-trisubstituted purine compound.
In one embodiment, this invention is A 2,6,9-trisubstituted purine composition of matter having the following formula: 
where
X is a amino, oxo, thio, or sulfone moiety;
R1 is halogen or R1xe2x80x2xe2x80x94X wherein X is an amino, oxo, thio, or sulfone moiety. X is preferably amino.
R1xe2x80x2 is a lower alkyl, substituted lower alkyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl, heterocycle, hetaryl, substituted hetaryl, aralkyl, heteroaralkyl, heteroalkyl, alkyl alkenyl, alkyl alkynyl, alkyl cycloalkyl, or alkyl cycloheteroalkyl, each having from 1 to 20 carbon atoms;
R2 is hydrogen, lower alkyl, substituted lower alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heterocycle, hetaryl, substituted hetaryl, aralkyl, heteroaralkyl, heteroalkyl, alkyl alkenyl, alkyl alkynyl, alkyl cycloalkyl, or alkyl cycloheteroalkyl;
R3 is halogen, hydroxyl, thio, alkoxy, alkylthio, lower alkyl, xe2x80x94NR4R5 or a component having the formula: 
xe2x80x83where m=1-3, n=1-3, and o=1-3; Y=carbonyl, xe2x80x94NR4R5, hydroxyl, thiol, alkoxy, alkythio, and wherein R4 and R5 are each (independently) selected from the group including hydrogen, lower alkyl, substituted lower alkyl, alkoxy, amino, amido, carboxyl, cycloalkyl, substituted cycloalkyl, heterocycle, cycloheteroalkyl, substituted cycloheteroalkyl, acyl, aryl, substituted aryl, aryloxy, hetaryl, substituted hetaryl, aralkyl, heteroaralkyl, alkyl alkenyl, alkyl alkynyl, alkyl cycloalkyl, alkyl cycloheteroalkyl, or cyano; having from 1 to 20 carbon atoms, and preferably form 2 to 6 carbon atoms. Furthermore, when Y is carbonyl, Rxe2x80x24 does not exist in the composition. R4xe2x80x3 and R5xe2x80x3 may be a single oxygen atom and R4xe2x80x2xe2x80x3 and R5xe2x80x2xe2x80x3 may be a single oxygen atom. R4 and R5 are preferably the same or different substituted lower alkyl having 2 to 6 carbon atoms and preferably CH2CH2OH, CH2HC(CH3)OH and mixtures thereof. There are some limitations to the scope of R1, R1xe2x80x2, R2, R3 when R3 is 2-hydroxyethylamino and R2 is methyl, R1xe2x80x2xe2x80x94X cannot be amino, 3-methyl-2-butenylamino, benzylamino, or 3-hydroxybenzylamino. When R3 is 2-hydroxyethylamino and R2 is isopropyl, R1xe2x80x2xe2x80x94X is not benzylamino, 3-hydroxybenzylamino, or 3-methylbutylamino. When R3 is 2-hydroxyethylamino and R2 is 2-hydroxyethyl, R1xe2x80x2xe2x80x94X cannot be benzylamino. When R3 is selected from the group consisting of 2-propanol-2-methylamino and 2-dimethylaminoethylamino and R2 is methyl, then R1xe2x80x2xe2x80x94X cannot be benzylamino.
In another embodiment, this invention is a method for inhibiting cell proliferation in mammals comprising administering a therapeutically effective amount of the composition of claim 1 to the mammal. The method is useful for treating cell proliferation disorders such at rheumatoid arthritis, lupus, type I diabetes, multiple sclerosis, cancer, restenosis, host graft disease, and gout.
In yet another embodiment, this invention is a pharmaceutical composition of matter comprising the composition above in an admixture with one or more pharmaceutical excipients.
In still another embodiment, this invention is a composition useful for treating fungal infections (fungi) in humans, animals, and in plants.